Phage display selection of efficient glutamine-donor substrate peptides for transglutaminase 2

Zsolt Keresztessy, Éva Csosz, Jolán Hársfalvi, Krisztián Csomós, Joe Gray, Robert N. Lightowlers, Jeremy H. Lakey, Zoltán Balajthy, László Fésüs

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

Understanding substrate specificity and identification of natural targets of transglutaminase 2 (TG2), the ubiquitous multifunctional cross-linking enzyme, which forms isopeptide bonds between protein-linked glutamine and lysine residues, is crucial in the elucidation of its physiological role. As a novel means of specificity analysis, we adapted the phage display technique to select glutamine-donor substrates from a random heptapeptide library via binding to recombinant TG2 and elution with a synthetic amine-donor substrate. Twenty-six Gln-containing sequences from the second and third biopanning rounds were susceptible for TG2-mediated incorporation of 5-(biotinamido)penthylamine, and the peptides GQQQTPY, GLQQASV, and WQTPMNS were modified most efficiently. A consensus around glutamines was established as pQX(P,T,S)l, which is consistent with identified substrates listed in the TRANSDAB database. Database searches showed that several proteins contain peptides similar to the phage-selected sequences, and the N-terminal glutamine-rich domain of SWI1/SNF1-related chromatin remodeling proteins was chosen for detailed analysis. MALDI/TOF and tandem mass spectrometry-based studies of a representative part of the domain, SGYGQQGQTPYYNQQSPHPQQQQPPYS (SnQ1), revealed that Q6, Q8, and Q22 are modified by TG2. Kinetic parameters of SnQ1 transamidation (KMapp = 250 μM, kcat = 18.3 sec-1, and kcat/KMapp = 73,200) classify it as an efficient TG2 substrate. Circular dichroism spectra indicated that SnQ1 has a random coil conformation, supporting its accessibility in the full-length parental protein. Added together, here we report a novel use of the phage display technology with great potential in transglutaminase research. Published by Cold Spring Harbor Laboratory Press.

Original languageEnglish
Pages (from-to)2466-2480
Number of pages15
JournalProtein Science
Volume15
Issue number11
DOIs
Publication statusPublished - 2006

Keywords

  • Chromatin remodeling proteins
  • Glutamine-donor substrate
  • Glutamine-rich
  • Phage display
  • Transglutaminase 2

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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